JP4488950B2 - Macrohomopsis gum high production bacteria selection method, and macrohomopsis gum production method using high production bacteria - Google Patents

Description

The present invention relates to a method for selecting or isolating cells (high-producing bacteria) having high β-glucan (macrohomopsis gum) -producing ability from microorganisms belonging to the genus Macrophomopsis . The present invention also relates to a method for defusing cells that can be used effectively in selecting the cells (high-producing bacteria).

Furthermore, the present invention relates to a method for producing β-glucan (macrohomopsis gum) using a microorganism belonging to the genus Macrophomopsis , and more particularly to a method for producing β-glucan (macrohomopsis gum) with high yield.

Conventionally, it is well known that microorganisms produce various β-glucans. For example, curdlan produced by microorganisms belonging to the genus Agrobacterium, dizoferran produced by basidiomycete Suehirotake (hereinafter, Non-Patent Document 1), macrohomopsis gum produced by microorganisms belonging to the genus Macrophomopsis ( Patent document 1) can be mentioned.
Of these, β-glucan other than dizoferran is approved for use as a food additive (Non-Patent Documents 2 to 3). For example, macrohomopsis gum is used as a thickening stabilizer. Moreover, the use as a cholesterol inhibitor is also known (patent document 2).

However, according to the culturing method described in Patent Document 1, for example, the amount of β-glucan (macrohomopsis gum) produced by macrohomopsis is as low as 1.5 g / L or less after 4 days of culturing. However, the fact is that they are not mass-produced.
"New developments in natural and biopolymer materials" CMC Publishing p84-89, p146-147 “Third Edition: Existing Additives Voluntary Standards” November 2002 Japan Food Additives Association p120 "Third edition, existing additives, voluntary standards" November 2002, Japan Food Additives Association p263 JP-A-4-122701 JP-A-6-135839

  As described above, macrohomopsis gum is low in β-glucan production ability of microorganisms belonging to the genus Macrohomopsis (hereinafter simply referred to as “macrohomopsis”), or low yield of conventional production methods. For the reasons mentioned above, the fact is that they are not mass-produced and are not used effectively.

  Therefore, an object of the present invention is to provide a production method for obtaining macrohomopsis gum in a high yield using a microorganism belonging to the genus Macrohomopsis. Furthermore, the present invention provides a method for selecting cells having high β-glucan (macrohomopsis gum) -producing ability (high-producing bacteria) from macrohomoposis, and a solution of the cells effectively used for the selection. An object is to provide a yarn method.

  As a result of intensive studies to solve the above problems, the present inventors have found that cells having at least two types of shapes exist in the cells belonging to the genus Macrohomopsis (macrohomopsis). There are differences in the production ability of β-glucan depending on the shape of the fungus body. I found that there was a "recessive bacterium"). And it confirmed that macrohomopsis gum could be produced with a high yield by using long filamentous microbial cells (dominant bacteria) with high β-glucan production ability and few branches as the inoculum.

  By the way, macrohomopsis is a filamentous fungus having a filamentous form having a plurality of branches, and forms a fungus group in which the mycelium of each fungus is complicatedly entangled and mixed (FIG. 1). In other words, in the group of macrohomopsis cells, the hyphae of the above dominant bacteria (long filamentous cells with few branches) and recessive bacteria (short filamentous cells with many branches) are intertwined in a complex manner. It is formed in a state. For this reason, in order to obtain an effective inoculum (dominant bacterium) for the production of the macrohomopsis gum, a method for selecting and collecting the dominant bacterium from the macrohomopsis cell group is required.

  Therefore, the inventors of the present invention have been researching with the aim of developing such a method. A mycelium is cut by homogenizing a solution containing macrohomopsis to prepare a cell suspension and then diluting it. It is possible to easily disentangle complex hyphae without inconvenience (such as unwinding), and as a result, from the obtained mycelia, by morphological observation, etc. -It was found that it can be selected as a glucan-producing bacterium.

  Furthermore, since the microbial cells (mycelium) are individually dispersed in the microbial cell suspension prepared by the above method, it is possible to obtain 1 by collecting a predetermined volume of the sample from the microbial cell suspension. It was found that only one cell (one hypha) can be obtained. Therefore, based on this knowledge, a sample of a predetermined volume is collected from the cell suspension, the β-glucan production ability of each sample is evaluated, and selection is performed using the production amount of β-glucan as an index. As a result, it was found that dominant bacteria can be easily selected and obtained for each microbial cell, and it was confirmed that such a method is effective as a method for selecting dominant bacterium (one microbial cell) instead of morphological observation.

The present invention is based on such findings and includes the following embodiments:
Item 1. A method of unwinding a group of cells belonging to the genus Macrophomopsis and formed by intertwining mycelia, homogenizing a solution containing the cells, and diluting the obtained cell suspension.

Item 2. A method for selecting or isolating long filamentous cells with few branches from a group of cells belonging to the genus Macrophomopsis having the following steps:
(a) a step of preparing a cell suspension by homogenizing a solution containing cells belonging to the genus Macrophomopsis (homogenization step),
(b) Collect a portion of the cell suspension obtained in the above step and dilute it at a rate that contains one cell (one mycelium) per predetermined volume (x mL). Preparation process (dilution process)
(c) A step of collecting a plurality of x mL samples from the diluted suspension, inoculating each into a medium, and culturing (culture step)
(d) a step (selection step) of selecting a culture having a high β-glucan production amount from the plurality of cultures obtained in (c),
(e) The culture selected in (d) or the culture obtained by culturing it again as a seed fungus is used as a solution containing cells belonging to the genus Macrophomopsis , and (a) to (d ) Step is repeated n times (n = 0 or a positive number of 1 or more),
(f) A step of obtaining a culture with a high β-glucan production amount selected in the (d) step of (n + 1) th time (n = 0 or a positive number of 1 or more).

Item 3. A method for producing macrohomopsis gum, comprising aeration culture of cells belonging to the genus Macrophomopsis in a medium, and collecting β-glucan produced in the culture, wherein the cells are used for the above-mentioned culture ( A method characterized by using long filamentous cells with few branches as inoculum.
Item 3-1. Item 4. A method according to Item 3, wherein a long filamentous cell having a small number of branches selected is used as the cell (inoculum) used for culture.
Item 3-2. Item 4. The method according to Item 3, wherein a long filamentous fungus body with few branches separated from a short filamentous shape with many branches is used as the fungus body (seed fungus) used for culture.
Item 3-3. Item 4. The method according to Item 3, wherein a long filamentous fungus culture with few branches is used as an inoculum for culture.
Item 4. The macrohomopsis gum according to Item 3, wherein the long filamentous fungus having few branches is selected or isolated by the method described in Item 2, or selected or isolated by shape observation. Production method.

Item 5. Macrohomopsis gum having the following properties obtained by the method according to Item 3 or 4;
(A) All of the main chain D-glucopyranosyl residues are β-1,3 bonded, and the ratio is one for every four D-glucopyranosyl residues in the main chain. -Neutral polysaccharides with one β-1,6-linked glucopyranosyl residue,
(B) The one having a weight fraction average molecular weight of 100,000 to 10 million Da.

  The microorganism belonging to the genus Macrohomopsis is a conventionally known incomplete filamentous fungus as described in, for example, Japanese Patent Application Laid-Open No. 1-63370 (trusted by Mikoken 9936). In the examples described later, the deposited bacteria belonging to the macrohomopsis (San-Ei-1 strain: the indication “Macrophomopsis San-Ei-1 San-Ei Gen FFI, 20050125” for the identification given by the depositor, the accession number “ FERM P-20389 ") is used as an example, but the method of the present invention is not limited to this deposited bacterium, and is a method applicable to all microorganisms belonging to the genus Macrophomopsis.

  Hereinafter, the present invention will be described in detail.

(1) Selection method of dominant bacteria As described above, according to the researches of the present inventors, macrohomopsis has a long filamentous fungus body with few branches (long filamentous fungus with few branches) and a short filamentous mold with many branches. There are two types of fungus bodies (short filamentous fungi with many branches). The former has high β-glucan production ability (dominant bacteria), the latter has low β-glucan production ability (recessive bacteria), and β-glucan production. It was found to have distinctly different properties in performance. For this reason, in order to produce β-glucan with high yield using macrohomopsis, long filamentous cells with few branches (long filamentous fungi with few branches) are selected from macrohomopsis cells. It is necessary to use as an inoculum. From this point of view, the present invention provides a method for selecting or isolating long filamentous fungi with few branches from a group of macrohomopsis cells in which at least two types of cells are mixed.

The method of the present invention comprises the following steps (a) to (f):
(a) a step of preparing a cell suspension by homogenizing a solution containing cells belonging to the genus Macrophomopsis (homogenization step),
(b) Collect a portion of the cell suspension obtained in the above step and dilute it at a rate that contains one cell (one mycelium) per predetermined volume (x mL). Preparation process (dilution process)
(c) A step of collecting a plurality of x mL samples from the diluted suspension, inoculating each into a medium, and culturing (culture step)
(d) a step (selection step) of selecting a culture having a high β-glucan production amount from the plurality of cultures obtained in (c),
(e) The culture selected in (d) or a culture obtained by culturing it again as a seed fungus is used as a solution containing cells belonging to the genus Macrophomopsis , and (a) to (d ) Step is repeated n times (n = 0 or a positive number of 1 or more),
(f) A step of obtaining a culture with a high β-glucan production amount selected in the (d) step of (n + 1) th time (n = 0 or a positive number of 1 or more).

  Among the above steps, the homogenization step (step (a)) and the dilution step (step (b)) correspond to a step of unwinding (unwinding) the complexly intertwined mycelium.

  The cell solution used in the homogenization step (step (a)) is not limited, but is preferably a solution containing cells at a concentration of 1 to 30% by weight. The preferred cell concentration is 1 to 15% by weight, more preferably 5 to 15% by weight.

  The solution for preparing such microbial cells may be sterilized without affecting the survival and growth of the microbial cells, and examples thereof include sterilized water and nutrient medium.

  Homogenization can be performed using a homogenizer, a stirrer, a disperser or the like conventionally used in the art. As homogenization conditions, it is desirable to set the rotation speed of the rotary blade or stirring blade of the homogenizer to 3000 to 30000 rpm, preferably 6000 to 15000 rpm, more preferably 8000 to 12000 rpm. If the rotational speed is extremely smaller than this, the intended sufficient effect cannot be obtained, and if the rotational speed is extremely higher than this, the mycelium is cut and one hypha cannot be obtained.

  The homogenization is desirably performed at 10 to 40 ° C., preferably 20 to 35 ° C., for 1 to 3600 seconds, preferably 10 to 900 seconds, more preferably 60 to 300 seconds.

  The bacterial cell-containing material thus obtained, that is, the bacterial cell suspension prepared by the homogenization is then subjected to the dilution step (b). The dilute solution used here may be sterilized as described above without affecting the survival and growth of the cells, and for example, the sterilized water or nutrient medium used in the above steps can be used in the same manner. .

  The dilution factor is 1 to several bacterial cells (1 to several mycelium), preferably 1 or 2 to 3 bacterial cells (1 or 2 to 1) in a predetermined volume (x mL) of the bacterial cell suspension obtained above. The ratio is not particularly limited as long as the ratio includes three mycelia). Ideally, it is preferable to dilute so that approximately 1 fungus body (almost 1 hyphae) is contained in a predetermined volume (x mL).

  Here, the predetermined volume is not particularly limited and can be arbitrarily determined, but from the viewpoint of handling, it is usually 0.01 to 0.05 mL, preferably 0.01 to 0.03 mL, more preferably 0.01 to 0.02 mL. can do.

  As the dilution factor, for example, the cell suspension obtained in the homogenization step (step (a)) has a cell concentration of 1 to 30% by weight, preferably 1 to 15% by weight, more preferably as described above. In the case of 5 to 15% by weight, the dilution factor can be 100 to 10,000 times, preferably 100 to 10,000 times, preferably 1000 to 10,000 times. In addition, after the dilution, stirring or shaking may be performed as necessary so that the bacterial cells are uniformly dispersed in the diluted suspension.

  As shown in the examples described later, the homogenization step (step (a)) and the dilution step (step (b)) unravel the intertwined hyphae of the macrohomopsis cell group and add them individually to the diluted suspension. Can be dispersed. The present invention includes a method for deflating macrohomopsis cells having such a homogenization step (step (a)) and a dilution step (step (b)).

  In addition, whether or not the mycelium of the macrohomopsis cell group was unwound in the homogenization step (step (a)), and the presence of the cell in a predetermined volume (x mL) of the cell suspension in the dilution step (step (b)) As a method for confirming whether the number is 1 to several microbial cells (preferably 1 to 3 microbial cells, more preferably 1 microbial cell), microscopic observation can be exemplified. Specifically, when a predetermined volume (0.01 mL) of a cell suspension prepared by dilution after homogenization is observed with an optical microscope and it is determined that the mycelium has not been unwound, homogenize again. If it is determined that there are many mycelia present, further dilute to increase the dilution rate. That is, the homogenization time in the homogenization step ((a) step) and the dilution rate in the dilution step ((b) step) can be set to the optimum conditions by appropriately adjusting by microscopic observation.

  By using the diluted suspension prepared in the above dilution step (step (b)), it is possible to select and obtain long filamentous fungi that are visually less branched from the macrohomopsis using a microscope or the like. However, by performing the above steps (c) to (f), it is also possible to select and obtain long filamentous fungi with little branching.

  Of steps (c) to (f), step (c) (culture step) and step (d) (selection step) are the β-glucan producing ability from the diluted suspension obtained in step (b). This is a series of steps for selecting cells (mycelium) having a high β as the index of β-glucan production.

  Specifically, the step (c) (culture step) includes macrohomopsis at a ratio of 1 to several fungi (1 to several mycelia) (preferably 1 to 3 fungi, more preferably approximately 1 fungus). In this step, a plurality of samples (xmL) are sampled, and each is inoculated into a nutrient medium and cultured.

  Here, as the nutrient medium, a known medium used for the production of β-glucan using macrohomopsis can be used (for example, see JP-A-4-127701). Usually, a medium containing a carbon source and a nitrogen source is used.

  Examples of the carbon source used in the nutrient medium include glucose, maltose, starch, starch degradation product, sucrose, fructose, lactose, molasses, glucose, lactose, fructose, galactose, melibiose, raffinose, stachyose or a mixture thereof. be able to. The concentration of such a carbon source in the nutrient medium is not particularly limited, but can be typically 2 to 20% by weight, preferably 2 to 5% by weight.

  As the nitrogen source to be blended in the nutrient medium, almost all organic or inorganic nitrogen sources used for culturing microorganisms can be used. Examples thereof include cottonseed flour (Pharmamedia), corn steep liquor, yeast extract, various peptones, oatmeal meat extract, casein hydrolyzate, ammonia salt, and nitrate. Absorbent cotton seed powder (Pharmamedia) is preferable. The concentration of the nitrogen source in the nutrient medium can be appropriately set according to the type of the nitrogen source, but when using, for example, cotton wool powder, usually 0.15-0.45% by weight, preferably 0.16-0.42% by weight, A concentration of 0.18 to 0.35% by weight, more preferably 0.2 to 0.3% by weight, can be exemplified.

  If necessary, the nutrient medium may contain, as other components, inorganic salts such as sodium chloride, magnesium, calcium, or phosphoric acid, and trace amounts of metal salts such as iron, copper, and manganese. You may mix | blend.

  The culture temperature is usually in the range of 10 to 40 ° C, preferably 20 to 35 ° C, more preferably 29 to 35 ° C. The pH of the culture solution should be in the range of 3.5 to 9, preferably 5 to 8. Depending on the situation, the pH may be adjusted with alkali salts such as sodium hydroxide, potassium hydroxide, ammonium nitrate, amino acids, or buffers. It may be adjusted to a certain level.

  The culture method is not particularly limited as long as it is aerated. For example, deep aeration agitation culture (culture by stirring a culture solution using a stirring blade), shaking culture, deep aeration culture, drum rotation culture, or the like is possible. A method can be mentioned. For example, the aeration conditions employed for the culture can be preferably 0.1 to 3 vvm, more preferably 0.5 to 2 vvm, and still more preferably 1 to 2 vvm. In addition, as gas used for ventilation | gas_flowing, the gas containing the sterilized oxygen, specifically, the air which filtered and sterilized by letting air pass through a filter can be illustrated.

  Preferably, the culture is performed with stirring under the above-described aeration conditions (deep aeration stirring culture method). The number of rotations of stirring (stirring speed of the stirring blade) is not particularly limited, but it is usually 50 to 300 rpm, preferably 100 to 250 rpm, more preferably 200 to 250 rpm.

  The culture time can be appropriately adjusted according to the culture conditions. Usually, it can be appropriately selected from the range of 3 to 6 days. For example, under the above temperature conditions, under aeration conditions of 1 to 2 vvm, in the case of deep aeration and agitation culture with a stirring blade stirring speed of 200 to 250 rpm, the culture is usually 3 to 6 days, preferably 4 to 5 days It is desirable to do it.

  Step (d) (selection step) is a step of selecting a culture having a high β-glucan production amount from the plurality of cultures prepared above. That is, it is a step of selecting cells (mycelium) having high β-glucan production ability using the amount of β-glucan produced in the culture as an index. The production amount of β-glucan in the culture can be evaluated by measuring the production amount of β-glucan contained in the liquid phase part obtained by solid-liquid separation of the culture. Specifically, the amount of β-glucan produced in the culture is evaluated by first heating the culture obtained in the culturing step (step (c)) at about 80 to 121 ° C. for 10 minutes or longer, and filtering or centrifuging. Solid-liquid separation is performed according to a conventional method such as separation, and then an appropriate precipitating agent, for example, an organic solvent such as ethanol, methanol, isopropanol, propanol, acetone, is added to the culture solution (filtrate or supernatant) obtained by solid-liquid separation. A method of adding 10 to 60% by weight, preferably about 10 to 30% by weight, and evaluating the weight of the removed precipitate as the weight of β-glucan can be mentioned.

  Thus, a culture having a high β-glucan production amount can be selected using the β-glucan production amount of the culture as an index. In some cases, the culture selected here may be a culture of long filamentous fungi with few branches. In this case, by acquiring this culture, long filamentous fungi with few branches were selected (corresponding to step (f) in the case of n = 0).

  In addition, in order to improve the selection accuracy of the bacterial cells, the culture selected in the above step (d) (selection step) (culture with a large amount of β-glucan production) is further added to the following steps (e) and (f ) Can also be used in the process.

(e) The culture selected in (d) or the culture obtained by culturing it again as a seed fungus is used as a solution containing cells belonging to the genus Macrophomopsis , and (a) to (d ) Step is repeated n more times (n = 1 or more positive number), or
(f) A step of obtaining a culture having a high β-glucan production amount selected in the (d) step of the (n + 1) th (n = 1 or more positive number).

  In the step (e), the number of times n, that is, the number of repetitions of the steps (a) to (d) is not particularly limited as long as it is 1 or more, but usually n = 3 or more, preferably n = 5 or more. More preferably, n = 7 times or more.

In step (e), the solution to be used in step (a) as a solution containing cells belonging to the genus Macrophomopsis may be the culture selected in (d) or selected in (d). It may be a culture obtained by culturing the cultured product again as an inoculum. For example, if the culture selected in step (d) has a small amount of cells to be used in step (a), the culture selected in step (d) is once cultured by the latter method. After increasing the body weight, it can be used in step (a).

  Thus, in the (n + 1) th (n = 1 or more positive number) selection step (step (d)), a culture with a high β-glucan production amount is selected using the β-glucan production amount of the culture as an index. Can do. The culture corresponds to a culture of long filamentous fungi with few branches. Therefore, by obtaining a culture with a large amount of β-glucan produced here, long filamentous fungi with few branches were selected (corresponding to step (f) in the case of a positive number of n = 1 or more). ).

  The long filamentous fungus having few branches is a dominant fungus having a high β-glucan-producing ability in macrohomoposis. Therefore, the long-branch filamentous fungus selected by the above method (the culture of the short-branched long filamentous fungus) is suitable as an inoculum having high β-glucan production ability in the production of β-glucan using macrohomopsis. Can be used. That is, the above method is also a method of selecting dominant bacteria having high β-glucan production ability from macrohomopsis.

(2) Method for Producing Macrohomopsis Gum The present invention relates to a method for producing macrohomopsis gum in a high yield using a microorganism belonging to the genus Macrohomopsis (macrohomopsis).

  The method of the present invention is characterized by using the above-mentioned long filamentous fungus as such macrohomopsis.

  Here, the long branching fungus belonging to macrohomopsis is a dominant bacterium characterized by high ability to produce β-glucan, and as described above, its unique shape can be obtained as an index. Moreover, it can also acquire according to the selection method of said (1), The acquisition method in particular is not restrict | limited.

  The method of the present invention can be carried out using a known method as a method for producing β-glucan using macrohomopsis, except that the long filamentous fungus with few branches is used as an inoculum (see, for example, JP-A-4-127701). Issue gazette). Specifically, it is actually performed by aeration culture of the above-mentioned long filamentous fungus in a medium and collecting the β-glucan produced in the culture solution.

  Here, as a carbon source to be mixed in the nutrient medium, for example, glucose, maltose, starch, starch degradation product, sucrose, fructose, lactose, molasses, glucose, lactose, fructose, galactose, melibiose, raffinose, stachyose or a mixture thereof Can be mentioned. Glucose is preferable. The concentration of such a carbon source in the nutrient medium is not particularly limited, but can be typically 2 to 20% by weight, preferably 2 to 10% by weight, and more preferably 2 to 5% by weight.

  As the nitrogen source to be blended in the nutrient medium, almost all organic or inorganic nitrogen sources used for culturing microorganisms can be used. Examples include cottonseed flour (Pharmamedia), corn steep liquor, yeast extract, various peptones, oatmeal meat extract, casein hydrolyzate, ammonia salt, nitrate, and the like. Absorbent cotton seed powder (Pharmamedia) is preferable. The concentration of the nitrogen source in the nutrient medium can be appropriately set according to the type of the nitrogen source. For example, when absorbent cottonseed powder is used, it is usually 0.15 to 0.45% by weight (about 0.014 to 0.043 in terms of nitrogen element). % By weight), preferably 0.16 to 0.42% by weight (about 0.015 to 0.040% by weight in terms of nitrogen element), more preferably 0.18 to 0.35% by weight (about 0.017 to 0.034% by weight in terms of nitrogen element), more preferably 0.2 to An example is 0.3% by weight (about 0.019 to 0.029% by weight in terms of nitrogen element).

  In addition, by culturing microorganisms belonging to the genus Macrohomopsis under such nitrogen source concentration conditions (for example, nitrogen element equivalent concentration conditions), the macrohomopsis β-glucan production ability can be significantly increased. Homopsis gum can be obtained in high yield.

  If necessary, the nutrient medium may contain, as other components, inorganic salts such as sodium chloride, magnesium, calcium, or phosphoric acid, and trace amounts of metal salts such as iron, copper, and manganese. You may mix | blend.

  Culturing is performed at 10 to 40 ° C., desirably 20 to 35 ° C., for 3 to 6 days, preferably 4 to 5 days. The pH of the culture solution should be in the range of 3.5-9, preferably 5-8. Depending on the situation, the pH may be adjusted with alkali salts such as sodium hydroxide, potassium hydroxide, ammonium nitrate, amino acids, or buffer solutions. It may be adjusted to a certain level.

  The culture method is not particularly limited as long as it is aerated. For example, deep aeration agitation culture (deep aeration culture using agitator blades), agitation culture (culture by agitator culture using agitator blades), shaking, Examples of the method include aeration culture, deep aeration culture, and drum rotation culture. For example, the aeration conditions employed for the culture can be preferably 0.1 to 3 vvm, more preferably 0.5 to 2 vvm, and still more preferably 1 to 2 vvm. Examples of the gas used for ventilation include sterilized air, specifically, air sterilized by filtration through the air through a filter.

  Preferably, the culture is performed with stirring under the above-described aeration conditions (deep aeration stirring culture method, stirring culture method). The rotational speed of stirring (stirring speed of the stirring blade) is not particularly limited, but is usually 50 to 300 rpm, preferably 100 to 250 rpm, more preferably 200 to 250 rpm.

  The culture temperature is usually in the range of 10 to 40 ° C, preferably 20 to 35 ° C, more preferably 29 to 35 ° C.

  The culture time can be appropriately adjusted according to the culture conditions. Usually, it can be appropriately selected from the range of 3 to 6 days. For example, under the above temperature conditions, under aeration conditions of 1 to 2 vvm, in the case of deep aeration and agitation culture with a stirring blade stirring speed of 200 to 250 rpm, the culture is usually 3 to 6 days, preferably 4 to 5 days It is desirable to do it.

  Thus, the target β-glucan (macrohomopsis gum) is produced in the culture by macrohomopsis.

  The β-glucan (macrohomopsis gum) targeted by the present invention has all the D-glucopyranosyl residues of the main chain linked by β-1,3 bonds as shown in the following formula, and the D- It is a neutral polysaccharide having a structure in which one D-glucopyranosyl residue is linked by β-1,6 as a side chain at the position of C6 at a ratio of one to four glucopyranosyl residues.

  The β-glucan (macrohomopsis gum) targeted by the present invention includes those having a weight fraction average molecular weight of 100,000 to 10 million Da, preferably 1 million to 10 million Da. The weight fraction average molecular weight can be determined by fractionating the molecular weight by size exclusion chromatography and measuring each fractionated molecular weight with a multi-angle light scattering detector, as shown in the Examples below. .

  Next, the method of the present invention is carried out by collecting the target β-glucan (macrohomopsis gum) from the culture prepared by the above method. The method of collecting β-glucan (macrohomopsis gum) from the culture can be performed by appropriately combining conventional operations used when isolating and purifying polysaccharides from the culture.

  For example, first sterilize the culture, then solid-liquid separate the culture according to conventional methods such as filtration or centrifugation, remove the microbial cells obtained as solids, and obtain a culture solution (filtrate, supernatant) .

  Here, the culture is preferably sterilized by a method of heat-treating the culture. As temperature conditions of heat processing, 70-150 degreeC, Preferably 100-125 degreeC can be mentioned. The heat treatment is preferably a heat treatment at 70 to 150 ° C. for 1 to 120 minutes, more preferably a heat treatment at 100 to 125 ° C. for 5 to 20 minutes. As specific heating conditions, a condition of 15 minutes at 105 ° C. or 10 minutes at 121 ° C. can be exemplified.

  By performing this heat treatment, the culture can be sterilized, and at the same time, the viscosity of the culture can be lowered, and the working efficiency of subsequent solid-liquid separation (filtration, centrifugation) can be improved. In addition, this heat treatment can improve the yield of β-glucan (macrohomopsis gum) recovered in the culture solution (filtrate, supernatant). The reason for this is not clear, but it is thought that β-glucan (macrohomopsis gum) adhering to the cell surface is easily released from the cell by heat treatment.

  Next, an appropriate precipitating agent, for example, an organic solvent such as ethanol, methanol, isopropanol, propanol, acetone, or the like is added to the culture solution (filtrate, supernatant) obtained above in an amount of about 10 to 60% by weight, preferably 10 to 30%. It is added at a ratio of wt% to precipitate β-glucan (macrohomopsis gum).

  At this time, low-molecular substances may also be precipitated together with β-glucan (macrohomopsis gum), but such contaminants should be removed by adjusting the concentration of this precipitant (organic solvent). Can do. Further, the precipitate is redissolved in water, and then solid-liquid separation such as filtration or centrifugation is repeated, or precipitation with the above precipitating agent is repeated to remove mixed insoluble or impurities, and β-glucan ( Macrohomopsis gum) can be purified and obtained. The obtained purified product can be dried by an appropriate drying method such as freeze drying, shelf drying, drum drying, spray drying and the like. Moreover, you may grind | pulverize as needed.

  The culture solution (supernatant, filtrate) obtained by the above solid-liquid separation can be purified by a method of desalting by ion exchange treatment and decoloring with activated carbon or the like as necessary.

According to the above method, cells (mycelia) (dominant bacteria) having a high ability to produce β-glucan (macrohomopsis gum ) can be selected from macrohomopsis. By using the dominant bacterium, it is possible to produce β-glucan (macrohomopsis gum ) with high yield using macrohomopsis, and mass production of macrohomopsis gum , which has been a conventional problem, becomes possible.

  Reference examples and examples will be described below in order to clarify the configuration and effects of the present invention. However, the present invention is not affected by these examples.

The macrohomopsis used in the following Examples is a strain belonging to the genus Macrohomopsis having the following properties (hereinafter referred to as “San-Ei-1 strain”).
[Reference example]

I. Sampling site Separated from soil in Odawara City, Kanagawa Prefecture.

II. Properties on various culture media
The characteristics of the culture on various media based on macroscopic and microscopic observation of the San-Ei-1 strain are as follows.

(1) Macroscopic observation
The growth pattern of San-Ei-1 strain at 25 ° C was examined.

(1-1) Tuabeck Dock Agar Medium Growth was relatively fast, and mycelial elongation was observed on the second day of culture. On the fifth day of culture, white fluff-like mycelium was proliferating. Mycelia grow densely throughout. Around the 12th day, the colony becomes about 5.5 cm, and the back surface of the central part of the colony turns yellowish brown. From around the 3rd week, the mycelium started to rise slightly from the center of the colony.

(1-2) Potato dextrose agar medium Growth is relatively fast, and the mycelium is white fluff-like. In the central part of the colony, the mycelium is not very proliferative and is very active in the peripheral part, the mycelium becomes dense and becomes donut-like. Furthermore, the mycelium extends from there, forming a somewhat sparse mycelial band around the donut, and forming a dense mycelial band around the tip. And the sparse ligaments also become dense, resulting in a larger donut-like colony. Around the 12th day, the colony is about 6.5-7.5 cm. The back of the central part of the colony turns yellowish brown. At about the third week, mycelia grow to the center of the colony, and the hyphae are entirely covered. Thereafter, the mycelium swelled while coloring in a dark green color slightly in the periphery from the central part of the colony.

(1-3) Malt extract agar medium
Compared with (1-1) and (1-2), the initial white fluff-like mycelium growth is slow. However, colony expansion is fast. From around the 5th day, the sparse and dense hyphae pattern is observed from the central part of the colony to the periphery. Around the 12th day, the colony becomes 8.5 cm. In the 3rd week, the pattern gradually fades in a form that is covered with mycelia.

(1-4) Corn Mill Medium Growth is extremely fast, and the mycelium grows white fluff-like mycelium like the potato dextrose agar medium. It is suitable for conidial fruit formation, and it quickly flourishes as conidial fruit grows and matures. Conidia and fruit are formed by being slightly buried in agar. It turns purple when cultured with 1% mert extra added.

(1-5) Automill agar medium It is almost the same behavior as corn mill medium.
(2) Morphology under a microscope The mycelium on an agar medium such as corn mill is white fluff-like, grows as if diving in the agar, has a branch, and has a diaphragm. The conidia and fruit formed on the agar medium are spherical with dark brown tea and have an opening. The opening has a single hole, which is at the center. The conidial fruit pattern branches unemployed, with a diaphragm only at the base, and has a cylindrical shape. All conidia-forming cells are irregular. The conidia are colorless and have a spindle shape without a diaphragm. The tip of the conidia is obtuse and the rear end is cut.
(3) Growth pH
It can grow at pH 3.5-9, but cannot grow below pH 2.5. The optimum pH for growth is 5-8.
(4) Growth temperature
It grows in the temperature range of 10-40 ° C, but cannot grow below 5 ° C or above 45 ° C. Growth at 35-40 ° C is as fast as growth at 28 ° C. It is mainly for the formation of white mycelium, and it is more suitable at 28 ° C or less for the formation and maturation of conidia. The optimum temperature for growth and conidia formation is 20-30 ° C.

  San-Ei-1 strains with the above taxonomic characteristics are in accordance with Saccardo's classification format, DEUTERO-MYCOTINA (Incomplete Mycomycetes), Coelomycetes (Inconidial Conidiomycetes), Sphaeropsidales (Spheropsid) In addition, it is classified into bacteria belonging to the genus Macrophhomopsis from the shape of conidia and colorless and spindle-shaped monospores [BCSutton, “The Coelomycetes Fungi Imperfecti with Pycnidia Acervuli and Stromata ”, press Commonwealth Mycological Institute England 1980].

  The San-Ei-1 strain was named “Macrophomosis San-Ei-1 San-Ei Gen FFI, Inc 20050125” (indication for identification) by the present applicant. The deposit number: FERM P-20389 has been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, which has an address in Tsukuba City 1-1-1, Higashi 1-1-1, Tsukuba, Japan. 16 Production No. 389).

(1) Selection of bacterial cells FIG. 1 shows the shape of the San-Ei-1 strain. As can be seen from this, macrohomopsis exists in a state where mycelia of a plurality of cells are entangled.

2 ml of a solution containing macrohomopsis (San-Ei-1 strain) (mixed solution of 1.2 mL of bacterial solution and 0.8 mL of glycerol) is added to nutrient medium (glucose 5% by weight, cotton wool flour 0.3% by weight, KH ₂ PO ₄ Inoculated into 100 mL of an aqueous medium containing 0.1% by weight and 0.3% by weight of MgSO ₄ · 7H ₂ O, pH 7), and cultured with shaking at 25 ° C. for 4 days. After completion of the culture, 1 mL of the bacterial cells were suspended in 9 mL of sterilized water (diluted 10 times), and homogenized for 90 seconds at 25 ° C. using a homogenizer (homogenizer, DIAX600: Heidorf). As homogenization conditions, the rotation speed of the rotary blade or stirring blade of the homogenizer is (1) set to 3000 rpm, (2) set to 8000 rpm, and (3) set to 20000 rpm. Adopted. As a result, the mycelium of each cell could be unwound to the desired degree under the condition (2) (Fig. 2), but the cell could not be sufficiently unraveled under the condition (1) ( It was found that under the condition (3) and (3), the mycelium of the cells was cut and mixed with the cells (dominant and recessive) (FIG. 4).

  Therefore, when the cells homogenized under the condition (2) were further diluted 1000 to 10,000 times with sterilized water, the mycelium could be completely unwound for each mycelium. When this cell suspension was observed with an optical microscope, it was found that (a) slender and less-branched mycelium (long filamentous cell with few branches) (Fig. 5) and (b) short-branched mycelium (many branches) It was confirmed that two types of cells (short filamentous cells) (FIG. 6) were present.

(2) Evaluation of β-glucan production ability (a) The long filamentous fungus body obtained above (hereinafter simply referred to as “long filamentous fungus body”) and (b) The short filamentous fungus body with many branches ( Hereinafter, β-glucan-producing ability was evaluated for “short filamentous fungi” and the two were compared.

Specifically, an aqueous medium containing nutrient medium (glucose 5% by weight, cotton wool powder 0.5% by weight, KH ₂ PO ₄ 0.1% by weight, and MgSO ₄ · 7H ₂ O 0.3% by weight in a culture container having a capacity of 300 mL. Each cell ((a), (b)) was inoculated into 100 mL of the culture medium, and precultured with shaking culture at 25 ° C. for 4 days (shaking number: 130 rpm) (preculture). Nutrient medium (5% by weight glucose, 0.2% by weight cotton wool powder, 0.1% by weight KH ₂ PO ₄ , and MgSO ₄ · 7H ₂ O 0.3 in a 10 liter jar fan mentor (manufactured by Mitsuwa Chemical Co., Ltd.) A total volume of the above preculture ((a), (b)) was inoculated into 5 L of an aqueous medium containing 7% by weight, pH 7), and cultured at 25 ° C. for 4 days under deep aeration (aeration 2 vvm, agitation) Speed 250 rpm, stirring blade shape: portal type). In addition, each microbial cell ((a), (b)) used what was pre-cultured for 4 days at 25 degreeC with 100 mL of the same nutrient medium beforehand (inoculate the whole amount of pre-cultured microbial cells). The nutrient medium prepared in advance was dispensed 5 L into a 10 L jar fan mentor and heat-sterilized at 121 ° C. for 15 minutes.

On day 4 from the culture, a part of the culture was collected and centrifuged, and the supernatant was collected. Isopropyl alcohol was added to this so that it might become 20 weight%, β-glucan was precipitated, and the obtained precipitate was dried in an oven at 85 ° C. for 3 hours. The weight was evaluated, and this was converted into the weight (g) per liter of the culture solution to obtain the β-glucan production amount (g / L) of each bacterial cell.
The results are shown in Table 1.

  From Table 1, macrohomopsis has different β-glucan-producing ability depending on its shape, and long-filamentous cells with few branches have high β-glucan-producing ability (dominant bacteria), while short-filamentous cells with many branches produce β-glucan. It can be seen that the potency is extremely low (recessive bacteria). Therefore, short-branched cells with many branches are unsuitable as cultures (seeds) for the production of β-glucan (macrohomopsis gum), and long-filamentous cells with few branches are replaced with β-glucan (macrohomopsis gum). ) It was found that β-glucan (macrohomopsis gum) can be obtained with a high yield by using it as a culture (seed) for production.

Selection of bacterial cells and evaluation of β-glucan production ability (1) 2 ml of a solution containing a macrohomopsis (San-Ei-1 strain) (mixed solution of 1.2 mL of bacterial solution and 0.8 mL of glycerol) was added to the nutrient medium ( Aqueous medium containing 5% glucose, 0.3% by weight cottonseed flour, 0.1% by weight KH ₂ PO ₄ and 0.3% by weight MgSO ₄ · 7H ₂ O, pH 7) Inoculated into 100 mL, at 25 ° C. for 4 days Cultured with shaking.

After completion of the culture, 1 mL of bacterial cells were suspended in 9 mL of sterilized water (diluted 10-fold), and homogenized with a homogenizer (homogenizer, DIAX600: manufactured by Heidorf) for 90 seconds at 25 ° C. and at a rotation speed of 8000 rpm. After homogenization, the obtained suspension was further diluted 10,000 times with sterile water and shaken well. A plurality of 0.01 mL samples were taken from this diluted suspension, dropped on a glass slide and observed with an optical microscope. On average, approximately 0.01 mycelia were present in 0.01 mL of the diluted suspension. It was confirmed that
Thus, the selection of dominant bacteria with high β-glucan production ability (long filamentous cells with few branches) and recessive bacteria with extremely low β-glucan production ability (short filamentous cells with many branches) It can be performed by collecting while confirming the shape with a microscope. However, this operation is very technical and is not versatile. Also, it takes time to observe and collect mycelia, and there is a high possibility that various germs will be mixed during the work.

  (2) The same effect is obtained, and as a general-purpose method with a low possibility of contamination, the findings of (1) above (almost one mycelium is present in a 0.01 ml diluted suspension) Based on the above, inoculate the diluted suspension directly into 100 mL of 30-sample flask medium (the above nutrient medium) in 0.01 ml increments, and evaluate the β-glucan production ability of individual cells contained in each diluted suspension The method to do was adopted.

  Specifically, 0.01 ml (30 samples) of the diluted suspension prepared by homogenization in (1) above and diluting was inoculated into 100 mL of flask medium (the above nutrient medium), respectively, at 25 ° C. for 4 days. Cultured with shaking. After completion of the culture, for each specimen, 2 ml of a portion of the culture was dispensed from each culture into a serum tube and temporarily refrigerated (preserved culture). The remaining culture (98 mL) was sterilized by heating (121 ° C., 10 minutes), the culture was centrifuged, and the cells were sterilized to obtain a supernatant. Isopropyl alcohol was added to the obtained supernatant so as to be 20% by weight to precipitate β-glucan, and a specimen in which a large amount of β-glucan was precipitated was examined (Table 2, number of selections: first time). As shown in Table 2, there was a difference in the amount of β-glucan produced in each sample. In addition, it is considered that the specimen in which the growth of the bacteria was not observed even after culturing for 4 days did not have one mycelium in the diluted suspension used for the inoculation.

  Next, a preservation culture of a specimen in which a large amount of β-glucan was precipitated (that is, a culture solution having a high β-glucan production amount) was used as an inoculum and cultured with shaking in the above nutrient medium at 25 ° C. for 4 days. After completion of the culture, 1 ml of the culture was added to 9 ml of sterilized water in the same manner as described above, and dispersed for 90 seconds at a rotation speed of 8000 rpm with a homogenizer (Hydorf, Homogenizer DIAX 600). After dispersion, the 10-fold diluted suspension was further diluted 10,000 times with sterilized water and shaken well. 0.01 ml was collected, dropped on a slide glass, and one mycelium was confirmed with an optical microscope, and 0.01 ml was inoculated into 30 specimens of flask medium (the above nutrient medium).

  This was cultured with shaking at 25 ° C. for 4 days. After completion of the culture, for each specimen, 2 ml of a portion of the culture was dispensed from each culture into a serum tube and temporarily refrigerated (preserved culture). The remaining bacterial solution (98 mL) was sterilized by heating and then centrifuged to remove the cells. To the supernatant thus obtained, isopropyl alcohol was added so as to be 20% by weight to precipitate polysaccharide β-glucan, and specimens in which β-glucan was precipitated were examined (Table 2, number of selection: 2). Second time). As shown in Table 2, the number of samples in which more β-glucan was precipitated than the first time (that is, samples with a high β-glucan production amount) increased as a result of the second selection of bacteria.

  For this reason, using a preserved culture of a specimen in which a large amount of β-glucan was precipitated (that is, a culture solution having a high β-glucan production amount) as a seed fungus, the above-described culture and selection of bacteria were repeated a total of 8 times. . By doing so, most of the 30 specimens were classified into specimens with high β-glucan precipitation (that is, cultures with high β-glucan production) (Table 2). When the culture solution which was “++++” among the following evaluations was observed with a microscope, all the cells had long and few mycelia (FIG. 7).

A culture container with a capacity of 300 mL for the final cells (selected cells) (cells (c)) and the first unselected cells (San-Ei-1 strain) (cells (d)) Each bacterial cell (((5% by weight glucose, 0.5% by weight of cottonseed cottonseed powder, 0.1% by weight of KH ₂ PO ₄ and 0.3% by weight of MgSO ₄ · 7H ₂ O)) c) and (d)) were inoculated and cultured with shaking at 25 ° C. for 4 days (number of shaking: 130 rpm). On day 4 from the culture, the culture was collected and centrifuged, and the supernatant was collected. Isopropyl alcohol was added to this so that it might become 20 weight%, β-glucan was precipitated, and the obtained precipitate was dried in an oven at 85 ° C. for 3 hours. The weight was measured, and this was converted to the weight (g) per liter of the culture solution to obtain the amount of β-glucan produced (g / L) of each cell.

  As a result, the amount of β-glucan produced by the selected bacteria was 1.91 g / L. On the other hand, the amount of β-glucan produced by the cells that had not been selected was 0.93 g / L, 1/2 of the above.

  Based on the above, the above-mentioned method is used to select the long-filamentous cells with few branches (the culture solution of the long-filamentous cells with few branches) as the culture (seed) for the production of β-glucan (macrohomopsis gum). It was found that β-glucan (macrohomopsis gum) can be obtained with a high yield.

  The β-glucan obtained above was identified as macrohomopsis gum from the following physicochemical data.

  (1) Constituent sugar was hydrolyzed with 2.5N trifluoroacetic acid for 8 hours, neutralized with 2.5N sodium hydroxide, and then analyzed by HPLC using shodex SUGAR SP0810 (manufactured by Shokotsu Trading Co., Ltd.). was gotten. From this, it was confirmed that the polysaccharide obtained was mainly composed of glucose.

  (2) The binding mode is β-1.3 glucanase (laminarinase: manufactured by Sigma-Aldrich) for about 4 hours, digested with heat at 90 ° C for 10 minutes, and then inactivated by shodex SUGAR SP0810 (Shokotsu Trading Co., Ltd.) Glucose and gentiobiose were obtained by HPLC analysis using The ratio of the two was approximately glucose: gentiobiose = 3: 1. The polysaccharide obtained from this has a main chain of D-glucopyranose residues and β-1.3 bonds, and the main chain of D-glucopyranose residues has a ratio of 1 per 4 residues. It was a polysaccharide β-glucan having a side chain of -1.6 bond.

  (3) Molecular weights were obtained by subjecting a TSKgel GMPw column (manufactured by Tosoh Corporation) to molecular weight fractionation by size exclusion chromatography on the column and measuring each fractionated molecular weight with a multi-angle light scattering detector. The obtained molecular weight was found to be 100,000 to 10 million Da in terms of weight fraction average molecular weight.

<Conditions for size exclusion chromatography>
Apparatus: High-performance liquid chromatography (manufactured by JASCO Corporation)
Column: TSKgel GMPW _XL (manufactured by Tosoh Corporation)
Column temperature: 25 ° C
Mobile phase: 50 mM NaNO ₃ +4 mM NaN ₃
Flow rate: 0.5ml / min
Injection volume: 100 μl
Sample concentration: 0.5 mg / mL
Pretreatment: Filtration through a 0.45 μm membrane filter.

<Conditions for multi-angle light scattering detector>
Equipment: Multi-angle light scattering detector DAWN-DSP (manufactured by Wyatt), differential refraction detector (manufactured by JASCO Corporation)
Detection temperature: 25 ° C.

It is a figure which shows the shape of the microorganisms (San-Ei-1 strain | stump | stock) which belong to a macrohomoposis genus. In Example 1 (1), it is a figure which shows the shape of the said strain | stump | stock after homogenizing the said San-Ei-1 stock | strain on the conditions of 8000 rpm (rotation speed). In Example 1 (1), it is a figure which shows the shape of the said strain | stump | stock after homogenizing the said San-Ei-1 stock | strain on conditions of 3000 rpm (rotation speed). In Example 1 (1), it is a figure which shows the shape of the said strain | stump | stock after homogenizing the said San-Ei-1 stock | strain on the conditions of 20000 rpm (rotation speed). Among the cells belonging to the genus Macrohomopsis, it shows the shape of long filamentous mycelium with few branches. Among the cells belonging to the genus Macrohomopsis, the shape of short filamentous mycelium with many branches is shown. It is a figure which shows the shape of the microbial cell obtained by the microbial cell selection of Example 2. FIG.

Claims (3)

A method of unwinding a group of cells formed by intertwining mycelia belonging to the genus Macrophomopsis , and homogenizing a solution containing the cells at a rate of 1 to 30% by weight at a rotational speed of 6000 to 15000 rpm. The method of diluting the obtained cell suspension 100 to 10,000 times. A method for selecting or isolating β-glucan high-producing bacteria from a group of cells belonging to the genus Macrophomopsis having the following steps:
(a) a step of preparing a cell suspension by homogenizing a solution containing cells belonging to the genus Macrophomopsis at a rate of 1 to 30% by weight at a rotation speed of 6000 to 15000 rpm;
(b) Collect a portion of the cell suspension obtained in the above step and dilute it 100 to 10000 times so that one cell (one mycelium) is contained per predetermined volume (x mL). Preparing a suspension,
(c) collecting a plurality of x mL samples from the diluted suspension, inoculating each into a medium, and culturing;
(d) a step of selecting a culture with a high β-glucan production amount from the plurality of cultures obtained in (c), (e) the culture selected in (d) or obtained by culturing it again as an inoculum. Using the obtained culture for a solution containing cells belonging to the genus Macrophomopsis , repeating the steps (a) to (d) n times (n = 0 or a positive number of 1 or more),
(f) A step of obtaining a culture with a high β-glucan production amount selected in the (d) step of (n + 1) th time (n = 0 or a positive number of 1 or more). A method for producing macrohomopsis gum, comprising aeration culture of cells belonging to the genus Macrophomopsis in a medium and collecting β-glucan produced in the culture, wherein the cells are used for the culture ( A method comprising using, as an inoculum), a β-glucan high-producing bacterium selected by the method according to claim 2.

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